The ultimate objective of this project is to define the mechanisms by which herpes simplex viruses (HSV) invade human cells to establish infection. One or both of the two serotypes of HSV infect a majority of people in all human populations and are the cause of a spectrum of diseases (recurrent oral and genital lesions, severe disseminated disease in newborn infants, encephalitis, and blindness resulting from corneal infection). Understanding the different pathways by which HSV infects different types of human cells can lead to new types of antiviral drugs that block virus entry into cells and to the design of HSV live vaccines that might induce immunity without infecting critical cells such as those of the nervous system. Studies supported by this grant have led to the identification of several cell surface proteins that serve as coreceptors with cell surface glycosaminoglycans (GAG) to mediate the entry of HSV-1, HSV-2 and two animal alphaherpesviruses [pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1)] into human cells. Different human cell types express different subsets of the co- receptors, any one of which appears to be sufficient with GAGs to mediate entry. An HSV ligand for the co-receptors was shown to be gD in at least two cases. Specific aims and approaches for the next period include: (1) use of engineered hybrid proteins for identification of the structural domains of three related co-receptors (HveB, HveC and Pvr- HveD) responsible for their different spectra of entry activity with the viruses listed above; (2) use of HSV mutants for identification of the HSV glycoproteins required for viral entry mediated by each protein co- receptor, given preliminary indications that viral requirements for entry depend on the protein co-receptor expressed by the cell; (3) use of molecular methods to detect mRNA and protein for assessment of the expression levels of human co-receptors for HSV entry in various human cells and tissues and identification of the coreceptors used for entry in selected cultured human cell types. The results obtained should define the determinants for entry of HSV-1 or HSV-2 into various human cell types and the extent to which virus strain and cell susceptibility to entry govern spread of infections in human beings.